Functional Study of Salix matsudanaSmERF B2-8 Gene in Response to Waterlogging Stress

doi:10.13560/j.cnki.biotech.bull.1985.2025-1134

Abstract

Abstract:

Objective This study aimed to elucidate the biological function of the SmERF B2-8 gene in Salix matsudana Koidz. in response to waterlogging stress and verify whether it is a key factor regulating the tolerance to waterlogging. Method The SmERF B2-8 gene in S. matsudana Koidz. was cloned and analyzed bioinformatically. RT-qPCR was performed to measure its expression in different Salix varieties under waterlogging at various time. The overexpressing vector of SmERF B2-8 was further constructed and transgenic Arabidopsis thaliana was obtained. The study on the hypoxic stress response of A. thaliana was conducted. Concurrently, a gene silencing vector was constructed to conduct VIGS experiments on S. matsudana Koidz. andthe biological function of the gene under waterlogging stress was systematically verified. Result The coding sequence (CDS) of the SmERF B2-8 gene in S. matsudana Koidz. is 948 bp in length. Analysis of the promoter region of SmERF B2-8 revealed the presence of oxygen-related cis-acting elements. RT-qPCR results showed that the expression of the SmERF B2-8 gene increased with prolonged waterlogging time, with significantly upregulated expression at 12 h compared to 0 and 4 h. Transgenic Arabidopsis experiments showed that, SmERF B2-8-overexpressing transgenic Arabidopsis exhibited better tolerance to hypoxia stress, compared to wild-type Arabidopsis. VIGS experimental results demonstrated that when the SmERF B2-8 gene was silenced in S. matsudana Koidz., the tolerance to waterlogging stress was significantly reduced compared to the control group. Conclusion The SmERF B2-8 gene promoter in S. matsudana Koidz. contains hypoxia-responsive cis-acting elements, and its gene expression is upregulated after waterlogging stress. SmERF B2-8 transgenic A. thaliana exhibits enhanced hypoxia tolerance, while silencing of this gene in S. matsudana Koidz. reduced its tolerance to waterlogging. SmERF B2-8 gene in S. matsudana Koidz. is a positive regulatory factor in response to waterlogging stress.

Key words: Salix matsudana Koidz., ERF transcription factor, vector construction, hypoxia stress, VIGS experiment

LI Meng-ru, QIAN Chao-nan, KAN Si-wei, LIU Guo-yuan, ZHANG Jian, CHEN Yan-hong. Functional Study of Salix matsudanaSmERF B2-8 Gene in Response to Waterlogging Stress[J]. Biotechnology Bulletin, 2026, 42(7): 204-213.

Figures/Tables 7

Table 1 Primers used in the study

引物名称 Primer name	上游引物 Forward sequence （5'-3'）	下游引物 Reverse sequence （5'-3'）
SmERF B2-8	TCGTTTCCTCGCACTACA	ACAAAACCACCGGGAAAA
pYL156-SmERF B2-8	TAAGGTTACCGAATTCC TGAGGAGTTAATCGCGT	CGAGACGCGTGAGCTCA CAAAACCACCGGGAAAA
pWM101-SmERF B2-8	TCGAGCTTTCGCGAGCTC GGTACCATGTGTGGCGGT	CTCACCATGGTGGCGACC GGTACCTTACAGAGGTGC
qPCR-SmERF B2-8	ATGTGTGGCGGTGCTATTTT	GGGCTCAGAAAGGAATCAAA
pYL156-SmPDS	AATCAATGGGCCATGCCCTG	GCGGAGAAGAGCGAAAGGAT

Fig. 1 PCR amplification and vector construction of the SmERF B2-8 geneA: PCR amplification products. B. Overexpression vectors. C. Silencing vectors

Fig. 2 Physicochemical properties analysis and protein structure predicting results of SmERF B2-8A: Secondary structure. B: Tertiary structure. C: Conserved structural domains. D: Transmembrane domains. E: Hydrophobicity/hydrophilicity

Fig. 3 Phylogenetic and promoter cis-acting element analysis of SmERF B2-8A: Phylogenetic tree. B: SmERF B2-8 protein homologous sequence alignment. C: SmERF B2-8 promoter cis-acting element analysis

Fig. 4 Relative expressions of the SmERF B2-8 gene in different willows varieties under submergence stress

Fig. 5 Comparison of phenotypic and physiological indicators between wild type (WT) and transgenic Arabidopsis overexpressing SmERF B2-8A: Phenotypic comparison between WT and SmERF B2-8 transgenic Arabidopsis under normoxic and hypoxic conditions. B: Root length under normal conditions. C: Fresh weight under normal conditions. D: Root length under hypoxic conditions. E: Fresh weight under hypoxic conditions. F: Measured MDA content

Fig. 6 Silencing of SmERF B2-8 reduces the tolerance of willow to flood stressA: Phenotypic differences before and after submergence. B: Relative expression levels of the transgenic plant SmERF B2-8. C: Fresh weight of the roots. D: Dry weight of the roots. E: content MDA

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